Isopentenyl diphosphate (IPP) is the central intermediate in the biosynthesis of isoprenoids in all organisms. In higher plants, the formation of IPP is compartmentalized. The mevalonate (MVA) pathway, the enzymes of which are localized to the cytosolic compartment, produces the precursor of triterpenes (sterols) and certain sesquiterpenes (Newman, J. D. & Chappell, J., Crit. Rev. Biochem. Mol. Biol., 34:95-106 [1999]). In plastids, the deoxyxylulose-5-phosphate (DXP) pathway operates to supply IPP for the synthesis of monoterpenes and diterpenes (Eisenreich, W. et al., Tetrahedron Lett., 38:3889-3892 [1997]; Eisenreich, W. et al., Proc. Natl. Acad. Sci. USA, 93:6431-6436 [1996]), several sequiterpenes (McCaskill, D. & Croteau, R., Planta, 197:49-56 [1995]), tetraterpenes (carotenoids), and the prenyl side-chains of chlorophyll and plastoquinone (Lichtenthaler, H. K. et al., FEBS Lett., 400:271-274 [1997]).
In addition, there are examples of cooperation between the cytosolic and plastidial pathways in the biosynthesis of stress-induced and constitutively emitted volatile terpenoids from a variety of plants (Piel, J. et al., Angew. Chem. Int. Ed., 37:2478-2481 [1998]), and constitutive sesquiterpenes of chamomile (Adam, K.-P. & Zapp, J., Phytochemistry, 48:953-959 [1998]). In mammals, where the DXP pathway is not known to operate, and in plants, the individual biosynthetic steps of the MVA pathway have been well-characterized (Goldstein, J. L. & Brown, M. S., Nature (London), 343:425-430 [1990]; Bach, T. J., Crit. Rev. Biochem. Mol. Biol., 34:107-122 [1999]). However, for the recently discovered DXP pathway, which also occurs in many eubacteria (Rohmer, M., Prog. Drug Res., 50:135-154 [1998]), the biosynthetic sequence leading to the formation of IPP is still incompletely defined (The FIGURE).
The initial step of the pathway involves a condensation of pyruvate (C2 and C3) with D-glyceraldehyde-3-phosphate (GAP) to yield 1-deoxy-D-xylulose-5-phosphate (Rohmer, M., Biochem. J., 295:517-524 [1993]; Broers, S. T. J., Ph.D. thesis, Eidgenossische Technische Hochschule, Zurich, Switzerland [1994]; Schwarz, M. K., Ph.D. thesis, Eidgenossische Technische Hochschule, Zurich, Switzerland [1994]; Rohmer, M. et al., J. Am. Chem. Soc., 118:2564-2566 [1996]). The enzyme which catalyzes this reaction belongs to a novel family of transketolases, and the corresponding gene has been isolated from Escherichia coli (Sprenger, G. A. et al., Proc. Natl. Acad. Sci. USA, 94:12857-12862 [1997]; Lois, L. M. et al., Proc. Natl. Acad. Sci. USA, 95:2105-2110 [1997]), peppermint (Lange, B. M. et al., Proc. Natl. Acad. Sci. USA, 95:2100-2104 [1998]) and pepper (Bouvier, F. et al., Plant Physiol., 117:1423-1431 [1998]). In the second step of this pathway, rearrangement and reduction of DXP yield 2-C-methyl-D-erythritol (MEP) (Duvold, T. et al., Tetrahedron Lett., 38:4769-4772 [1997]; Duvold, T. et al., Tetrahedron Lett., 38:6181-6184 [1997]; Sagner, S. et al., Tetrahedron Lett., 39:2091-2094 [1998]) (The FIGURE). Recently, genes encoding this DXP reductoisomerase (DXR) have been cloned from E. coli (Takahashi, S. et al., Proc. Natl. Acad. Sci. USA, 95:9879-9884 [1998]), peppermint (Lange, B. M. & Croteau R., Arch. Biochem. Biophys., 365:170-174 [1999]), and Arabidopsis thaliana (Lange, B. M. & Croteau R., Arch. Biochem. Biophys., 365:170-174 [1999]; Schwender, J. et al., FEBS Lett., 455:140-144 [1999]). To date, no other intermediates on the route to IPP, the terminal product of the DXP pathway (McCaskill, D. & Croteau R., Tetrahedron Lett., 40:653-656 [1999]; Arigoni, D. et al., Proc. Natl. Acad. Sci. USA, 96:1309-1314 [1999]), have been identified.
As disclosed herein, sequencing of 1300 anonymous clones (expressed sequence tags, ESTs) from a cDNA library constructed from mRNA isolated from the oil gland secretory cells of peppermint (Mentha x piperita) (McCaskill, D. & Croteau, R., Planta, 197:49-56 [1995]), afforded, after extensive database comparisons, two clones having homologues of unknown function in plants and eubacteria, the sequences of which contained a motif with homology to the putative ATP-binding domain of the GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family of metabolite kinases. This putative kinase gene from peppermint and its E. coli orthologue, when overexpressed in E. coli, yielded a recombinant enzyme that catalyzes the ATP-dependent phosphorylation of isopentenol monophosphate (IP) to IPP. Feeding experiments with IP and several other isoprenoid precursors, using isolated peppermint secretory cells, confirmed the phosphorylation of IP to IPP to be the last step in the DXP pathway.